This invention relates to plant genes useful for the genetic manipulation of plant characteristics. More specifically, the invention relates to the identification, isolation and introduction of genes useful, for example, for altering the seed oil content, seed size, flowering and/or generation time, or vegetative growth of commercial or crop plants.
Through a coordination of the light and dark reactions of photosynthesis, plants assimilate CO2 in the formation of sugars. Via the catabolic and anabolic reactions of metabolism, these sugars are the basis of plant growth, and ultimately plant productivity. In the process of plant growth, respiration, which involves the consumption of O2 and catabolism of sugar or other substrates to produce CO2, plays a central role in providing a source of energy, reducing equivalents and an array of intermediates (carbon skeletons) as the building blocks for many essential biosynthesic processes. It is known that any two plants with equal photosynthetic rates often differ in both total biomass production and harvestable product. Therefore, the relationship between rate of respiration and crop productivity has been one of the most intensively studied topics in plant physiology. In a biochemical sense, respiration can be taken to be composed of glycolysis, the oxidative pentose phosphate pathway, the Kreb""s (tricarboxylic acid, TCA) cycle and the mitochondrial electron transport system. The intermediate products of respiration are necessary for growth in meristematic tissues, maintenance of existing phytomass, uptake of nutrients, and intra- and inter-cellular transport of organic and inorganic materials. In soybean there is evidence that an increase in respiration rate by the pod can lead to an increase in seed growth (Sinclair et al., 1987), while decreased respiration can result in decreased reproductive growth (Gale, 1974). Respiration is therefore important to both anabolic and catabolic phases of metabolism.
Although the pathways of carbon metabolism in plant cells are quite well known, control of the flux of carbon through these pathways in vivo is poorly understood at present. The mitochondrial pyruvate dehydrogenase complex (mtPDC), which catalyzes the oxidative decarboxylation of pyruvate to give acetyl CoA, is the primary entry point of carbohydrates into the Krebs cycle. The mtPDC complex links glycolytic carbon metabolism with the Krebs cycle, and, because of the irreversible nature of this reaction, the pyruvate dehydrogenase complex (PDC) is a particularly important site for regulation.
Mitochondrial PDC has been studied intensively in mammalian systems, and available knowledge about the molecular structure of plant mtPDC is largely based on studies of the mammalian mtPDC. The mtPDC contains the enzymes E1 (EC 1.2.4.1), E2 (EC 2.3.1.12) and E3 (EC 1.8.1.4) and their associated prosthetic groups, thiamine PPi, lipoic acid, and FAD, respectively. The E1 and E3 components are arranged around a core of E2. The E2 and E3 components are single polypeptide chains. In contrast, the E1 enzyme consists of two subunits, E1xcex1 and E1xcex2. Their precise roles remain unclear. Another subunit, the E3-binding protein, is thought to play a role in attaching E3 to the E2 core. The E1 kinase and phosphatase are associated regulatory subunits (Grof et al., 1995).
Plants are unique in having PDH complexes in two isoforms, one located in the mitochondrial matrix as in other eukaryotic cells, and another located in the chloroplast or plastid stroma (Randall et al., 1989). Although both plastidial and mitochondrial PDH complex isoforms are very sensitive to product feedback regulation, only the mitochondrial PDH complex is regulated through inactivation/reactivation by reversible phosphorylation/dephosphorylation (Miernyk and Randall, 1987; Gernel and Randall, 1992; Grof et al., 1995). More specifically, the activity of mitochondrial PDC (mtPDC) is regulated through product feedback inhibition (NADH and acetyl-CoA) and the phosphorylation state of mtPDC is determined by the combined action of reversible phosphorylation of the E1xcex1 subunit by PDC kinase (PDCK) and its dephosphorylation by PDC phosphatase. PDCK phosphorylates and inactives PDC, while PDC phosphatase dephosphorylates and reactivates the complex. Maximum PDC activity also appears to vary developmentally, with the highest catalytic activity observed during seed germination and early seedling development (e.g. in post-germinative cotyledons, Hill et al., 1992; Grof et al., 1995).
Acetyl-CoA, the product of PDC, is also the primary substrate for fatty acid synthesis. While it is known that plant fatty acid biosynthesis occurs in plastids has been the subject of much speculation. It remains a major question which has not been resolved. Because of the central role of acetyl-CoA in many metabolic pathways, it is likely that more than one pathway could contribute to maintaining the acetyl-CoA pool (Ohlrogge and Browse, 1995).
One school of thought takes the view that carbon for fatty acid synthesis is derived directly from the products of photosynthesis. In this scenario, 3-phosphoglycerate (3-PGA) would give rise to pyruvate, which would be converted to acetyl-CoA by pyruvate dehydrogenase in plastids (Liedvogel, 1986). This hypothesis has many appealing aspects, but also several unaddressed questions: (1) fatty acid synthesis occurs in photosynthetic (chloroplasts) and non-photosynthetic plastids (in root, developing embryo cotyledons, endosperm leucoplasts); (2) some plastids may lack 3-phosphoglycerate mutase (Kleinig and Liedvogel, 1980), an essential enzyme for converting 3-PGA, the immediate product of CO2 fixation, to pyruvate. (3) Acetate is the preferred substrate for fatty acid synthesis using isolated intact plastids, and there is evidence that a multienzyme system including acetyl-CoA synthetase and acetyl-CoA carboxylase, exists in plastids, which channels acetate into lipids (Roughan and Ohlrogge, 1996). It is almost certain that at least some of the acetyl-CoA in plastids is formed by plastidic pyruvate dehydrogenase, using pyruvate imported from the cytosol or produced locally by plastidial glycolysis.
A further possibility, especially in non-photosynthetic tissues (e.g., roots and developing embryos), is that acetyl-CoA, generated in the mitochondria, is an alternate means to provide acetate moieties for fatty acid synthesis (Ohlrogge and Browse, 1995). Mitochondrially-generated acetyl-CoA could be hydrolysized to yield free acetate, which could move into the plastid for conversion to acetyl-CoA via plastidial acetyl-CoA synthetase, an enzyme with 5- to 15-fold higher activity than the in vivo rate of fatty acid synthesis (Roughan and Ohlrogge, 1994). Alternatively, the mitochondrial acetyl-CoA could be converted to acetylcarnitine and transported directly into the plastid. Hence, in theory, the mitochondrial pyruvate dehydrogenase complex has an important role to play in fatty acid biosynthesis (see FIG. 1 of the accompanying drawings). The proof of this hypothesis has been hindered by the difficulties of directly measuring the existence of acetate in the cytosol.
The mitochondrial PDC (mtPDC) is a tightly regulated multiple subunit complex. As mentioned previously, one of the key regulatory components of this complex is PDH kinase (PDHK). PDHK functions as a negative regulator by inactivating PDH via phosphorylation. By modulating the PDCK, the activity of PDC can be genetically engineered.
Various attempts have been made to increase or channel additional carbon towards fatty acid biosynthesis. Targets have included genetically modifying acetyl-CoA carboxylase and pyruvate kinase gene expression through over-expression and antisense mRNA techniques with limited or no success.
However, there are many examples of successful modifications to plant metabolism that have been achieved by genetic engineering to transfer new genes or to alter the expression of existing genes, in plants. It is now routinely possible to introduce genes into many plant species of agronomic significance to improve crop performance (e.g. seed oil or tuber starch content/composition; meal improvement; herbicide, disease or insect resistance; heavy metal tolerance etc.) (Somerville, 1993; Kishore and Somerville, 1993; MacKenzie and Jain, 1997).
For example, increases in the proportions of some strategic fatty acids and in the quantities of seed oil have been achieved by the introduction of various fatty acid biosynthesis and acyltransferase genes in oilseed crops. These include the following demonstrations: Expression of an anti-sense construct to the stearoyl-ACP xcex949 desaturase in Brassicaceae led to an increase in the stearic acid content (Knutzon et al., 1992). Expression of a medium chain fatty acyl-ACP thioesterase from California Bay, in Brassicaceae was demonstrated to increase the lauric acid (12:0) content (Voelker et al., 1992; 1996). Expression of a Jojoba xcex2 keto-acyl-CoA synthase in low erucic acid Brassicaceae led to an increase the level of erucic acid (22:1); the effect following expression in high erucic acid cultivars was negligible (Lassner et al., 1996). Increased proportions of oleic acid in Brassica napus and in soybean have been achieved by silencing the microsomal FAD2 (xcex9412) desaturase (Hitz et al., 1995; Kinney, 1995; 1997). Transformation of Arabidopsis thaliana and rapeseed (B. napus) with a yeast sn-2 acyltransferase resulted in seed oils with increased proportions of 22:1 and other very long-chain fatty acids and significant increases in seed oil content (Zou et al., 1997).
Starch deposition has also been altered by genetic engineering. By expression of a mutant E. coli glgC16 gene encoding an ADP glucose pyrophosphorylase in potato tubers, an increase in starch accumulation was achieved (Stark et al., 1992).
However, because a PDHK gene has not heretofore been cloned from any plant, until now, no genetic modifications have addressed the possibility of altering carbon flux, increasing fatty acid synthesis, oil content or seed size, altering flowering and/or generation time, vegetative growth, or plant respiration/productivity by modulating plant mitochondrial PDH activity.
An object of the invention is to identify, isolate and characterize a pyruvate dehydrogenase kinase (PDHK) (gene and cDNA) sequence from Arabidopsis and to utilize this sequence in the genetic manipulation of plants.
Another object of the invention is to provide a vector containing the full-length PDHK sequence or a significant portion of the PDHK sequence from Arabidopsis, in an anti-sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants.
Another object of the invention is to provide a method to construct a vector containing the full-length PDHK sequence or a significant portion of the PDHK sequence from Arabidopsis, in a sense orientation under control of either a constitutive or a seed-specific promoter, for re-introducing into Arabidopsis or for introducing into other plants.
Another object of the invention is to provide a method of modifiying Arabidopsis and other plants to change their seed oil content.
Another object of the invention is to provide a method of modifiying Arabidopsis and other plants to change their average seed weight or size.
Another object of the invention is to provide a method of modifiying Arabidopsis and other plants to change their respiration rate during development.
Another object of the invention is to provide a method of modifiying Arabidopsis and other plants to change their vegetative growth characteristics.
Another object of the invention is to provide a method of modifiying Arabidopsis and other plants to change their flowering time or patterns of generative growth.
Yet another object of the invention is to provide a method of modifiying Arabidopsis and other plants to change the period required to reach seed maturity.
According to one aspect of the present invention, there is provided isolated and purified deoxyribonucleic acid (DNA) of SEQ ID NO:1 (pYA5; ATCC No 209562).
According to yet another object of the invention, there is provided a vector containing SEQ ID NO:1 or a part thereof, for introduction of the gene, in an anti-sense orientation (e.g. pAsYA5; ATCC No 209561) into a plant cell, and a method for preparing a vector containing SEQ ID NO:1 or a part thereof, for introduction of the gene in a sense orientation, into a plant cell.
The invention also relates to transgenic plants and plant seeds having a genome containing an introduced DNA sequence of SEQ ID NO:1 and a method of producing such plants and plant seeds.
The invention also relates to substantially homologous DNA sequences from plants with deduced amino acid sequences of 25% or greater identity, and 50% or greater similarity, isolated and/or characterized by known methods using the sequence information of SEQ ID NO:1, as will be appreciated by persons skilled in the art, and to parts of reduced length that are still able to function as inhibitors of gene expression by use in an anti-sense or co-suppression (Transwitch; Jorgensen and Napoli 1994) application. It will be appreciated by persons skilled in the art that small changes in the identities of nucleotides in a specific gene sequence may result in reduced or enhanced effectiveness of the genes and that, in some applications (e.g. anti-sense or co-suppression), partial sequences often work as effectively as full length versions. The ways in which the gene sequence can be varied or shortened are well known to persons skilled in the art, as are ways of testing the effectiveness of the altered genes. All such variations of the genes are therefore claimed as part of the present invention.
Stated more generally, the present invention relates to the isolation, purification and characterization of a mitochondrial pyruvate dehydrogenase kinase (PDHK) gene from the Brassicaceae (specifically Arabidopsis thaliana) and demonstrates its utility in regulating fatty acid synthesis, seed oil content, seed size/weight, flowering time, vegetative growth, respiration rate and generation time. Until now, no concrete data is available on the gene structure of plant PDC regulatory subunits (PDCK and PDC phosphatase).
The PDHK gene was cloned and characterized in the course of experiments designed to complement an E. coli mutant, JC201 (Coleman, 1990) with a plant (A. thaliana) cDNA library. By expressing the cDNA as a fusion protein in E. coli, its function was established as a PDHK in a protein kinase assay where it specifically phosphorylated the mammalian PDH E1xcex1/E1xcex2 subunits (the specific substrates of PDHK). The A. thaliana PDHK structure is significantly homologous to its mammalian counterpart, particularly among the functional domains.
The PDHK of the invention is useful in manipulating PDH activity, and the respiration rate in plants. For example, by transforming plants with a construct containing the partial PDHK gene in an antisense or in a sense orientation, under the control of either constitutive or tissue-specific promoters, the expression of mitochondrial PDHK can be silenced to some degree by anti-sense or co-suppression (Transwitch) phenomena (De Lange et al., 1995; Mol et al., 1990; Jorgensen and Napoli, 1994; Kinney, 1995), respectively. This can result in increased mitochondrial PDH activity, and hence an increased production or availability of mitochondrially-generated acetyl-CoA, or an increased respiration rate.
Alternatively, by over-expressing the full-length PDHK gene selectively in a tissue-specific manner, the activity of mitochondrial PDH may be negatively regulated, resulting in decreased respiratory rates in tissues, such as leaves or tubers, to decrease maintenance respiration and thereby increase the accumulation of biomass.
Some of the manipulations and deliverables which are possible using the PDHK gene or a part thereof, include, but are not limited to, the following: seeds with increased or decreased fatty acid and oil content; plants exhibiting early or delayed flowering times (measured in terms of days after planting or sowing seed); plants with increased or decreased vegetative growth (biomass); plants with root systems better able to withstand low soil temperatures or frost; plants with tissues exhibiting higher or lower rates of respiration; plants exhibiting an enhanced capacity to accumulate storage compounds in other storage organs (e.g. tubers); plants exhibiting an enhanced capacity to accumulate biopolymers which rely on acetyl moieties as precursors, such a polyhydroxyalkanoic acids or polyhydroxybutyric acids (Padgette et al., 1997).